LOCUS       sequence-399140-        6387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses recruitable fluorescent protein-tagged enzyme.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6387)
  AUTHORS   Wills RC, Doyle CP, Zewe JP, Pacheco J, Hansen SD, Hammond GRV
  TITLE     A novel homeostatic mechanism tunes PI(4,5)P2-dependent signaling at
            the plasma membrane.
  JOURNAL   J Cell Sci. 2023 Aug 3:jcs.261494. doi: 10.1242/jcs.261494.
  PUBMED    37534432
REFERENCE   2  (bases 1 to 6387)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell Sci.
            2023 Aug 3:jcs.261494. doi: 10.1242/jcs.261494."
COMMENT     Sequence Label: TagBFP2-FKBP-INPP5ED477N
FEATURES             Location/Qualifiers
     source          1..6387
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             44..838
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             44..838
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(98..117)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     708..727
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(1026..1045)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1070..1117
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1446..2034
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1935..1954
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        2209..2512
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2513..2716
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2667..2687
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2755..2764
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2761..3462
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIK
                     VVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMAL
                     KLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARY
                     CDLPSKLGHKLN"
     CDS             join(3520..3522,3523..3843)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     polyA_signal    5323..5444
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5360..5379)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5414..5433
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(5451..5906)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5588..5609)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5800..5819
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5933..6037
                     /gene="bla"
                     /label=AmpR promoter
     promoter        join(6039..6387,1..9)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        6039..6230
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(6062..6082)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6247..6382
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
       61 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
      121 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
      181 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
      241 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
      301 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
      361 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
      421 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
      481 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
      541 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
      601 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
      661 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
      721 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
      781 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
      841 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
      901 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
      961 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     1021 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     1081 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     1141 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     1201 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     1261 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     1321 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1381 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1441 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1501 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1561 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     1621 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1681 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1741 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     1801 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     1861 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     1921 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     1981 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2041 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2101 cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta gttattaata
     2161 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
     2221 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
     2281 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta
     2341 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc
     2401 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
     2461 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg
     2521 gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
     2581 ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
     2641 atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt
     2701 ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc ggtcgccacc
     2761 atggtgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg
     2821 gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag
     2881 accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct
     2941 actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc
     3001 ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg
     3061 ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc
     3121 aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc
     3181 tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac
     3241 atggccctga agctcgtggg cgggagccat ctgatcgcaa acgccaagac cacatataga
     3301 tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg
     3361 gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc
     3421 agatactgcg acctccctag caaactgggg cacaagctta attccggact cagatctcga
     3481 agcgcggccg cgggagcagg aggagcagct cgagcggcga tgggagtgca ggtggaaacc
     3541 atctccccag gagacgggcg caccttcccc aagcgcggcc agacctgcgt ggtgcactac
     3601 accgggatgc ttgaagatgg aaagaaattt gattcctccc gggacagaaa caagcccttt
     3661 aagtttatgc taggcaagca ggaggtgatc cgaggctggg aagaaggggt tgcccagatg
     3721 agtgtgggtc agagagccaa actgactata tctccagatt atgcctatgg tgccactggg
     3781 cacccaggca tcatcccacc acatgccact ctcgtcttcg atgtggagct tctaaaactg
     3841 gaagctcgag gcgcagcagc gggagcagga ggagcaggtc gatcggatct tgcagactac
     3901 aagctccgcg cgcagccgct cctggtgcgg gcccacagca gcctgggccc cggccggccg
     3961 cggagccccc tggcctgcga cgactgttcc cttcgctcgg ccaaatcctc cttcagcctc
     4021 ctggcgccca tccgcagcaa ggacgtccgc agcaggagtt acctggaggg cagcctcctg
     4081 gccagcgggg ccctgttggg ggcggatgag ctggcccgct acttcccaga ccggaacgtg
     4141 gcactcttcg tggccacctg gaacatgcag ggccagaagg agctcccgcc cagcctggac
     4201 gagttcctgc tcccagccga ggccgactat gcccaggacc tgtatgtcat cggggtccag
     4261 gagggctgtt ctgacaggcg ggagtgggag actcgtctgc aggagacgct gggcccgcac
     4321 tatgtgctgc tgtcctcggc ggcccacggc gtgctctaca tgtcgctctt catccgcagg
     4381 gacctcatct ggttctgctc agaggtggag tgctccacgg tgaccacacg catcgtgtct
     4441 cagatcaaga ccaagggggc cttgggcatc agcttcacct tttttggcac ttccttcctc
     4501 ttcatcacgt cccacttcac ctcaggtgac gggaaggtgg cggagcggct gctggactac
     4561 accaggactg tacaagccct ggtcctgccc agaaatgtgc ccgacaccaa cccctatcgc
     4621 tccagcgcag cggacgtcac cacccgcttc gatgaggtgt tctggtttgg aaacttcaac
     4681 ttccgcctga gtggcgggcg cacagtcgtg gacgccctcc tgtgccaggg cctggtggtg
     4741 gacgtgccgg cgctgctgca gcacgaccag ctcatccggg agatgcggaa agggtccatc
     4801 ttcaagggct tccaggagcc ggacatccac ttcctcccat catacaagtt tgacatcggg
     4861 aaggacacgt acgacagcac ctccaagcag aggacgccct catacacgga ccgcgtcttg
     4921 tacagaagcc gccacaaggg tgacatctgt cctgtgagct actcttcctg ccccgggatc
     4981 aagacgtccg accaccgccc tgtgtatggc ctcttccggg tgaaagtgag gccggggcga
     5041 gacaacattc cgttggcagc tggcaaattt gatagagaac tgtacttact aggaattaaa
     5101 agacggattt cgaaggagat tcagaggcag caagcactac agagtcagaa ctccagcacc
     5161 atcgcctccg tttcttgagg taccgcgggc ccgggatcca ccggatctag ataactgatc
     5221 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     5281 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     5341 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5401 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaacgcgt aaattgtaag
     5461 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     5521 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     5581 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     5641 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     5701 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     5761 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     5821 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     5881 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     5941 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     6001 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     6061 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     6121 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     6181 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     6241 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     6301 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     6361 tgaggaggct tttttggagg cctaggc
//
